Provitamin A (β-carotene) deficiency represents a very serious health problem leading to severe clinical symptoms in the part of the worlds population living on grains such as rice as the major or almost only staple food. In south-east Asia alone, it is estimated that 5 million children develop the eye disease xerophthalmia every year, of which 0.25 million eventually go blind (Sommer, 1988; Grant, 1991). Furthermore, although vitamin A deficiency is not a proximal determinant of death, it is correlated with an increased susceptibility to potential fatal afflictions such as diarrhoea, respiratory diseases and childhood diseases, such as measles (Grant, 1991). According to statistics compiled by UNICEF, improved provitamin nutrition could prevent 1-2 million deaths annually among children aged 1-4 years, and an additional 0.25-0.5 million deaths during later childhood (Humphrey et al., 1992). For these reasons it is very desirable to raise the carotenoid levels in staple foods. Moreover, carotenoids are known to assist in the prevention of several sorts of cancer and the role of lutein and zeaxanthin in the retina preventing macula degeneration is established (see e.g. Brown et al., 1998; Schalch, 1992).
Furthermore, carotenoids have a wide range of applications as colorants in human food and animal feed as well as in pharmaceuticals. In addition there is increasing interest in carotenoids as nutriceutical compounds in “functional food”. This is because some carotenoids, e.g. β-carotene, exhibit provitamin-A character in mammals.
Carotenoids are 40-carbon (C40) isoprenoids formed by condensation of eight isoprene units derived from the biosynthetic precursor isopentenyl diphosphate (see FIG. 1). By nomenclature, carotenoids fall into two classes, namely carotenes, comprising hydrocarbons whereas oxygenated derivatives are referred to as xanthophylls. Their essential function in plants is to protect against photo-oxidative damage in the photosynthetic apparatus of plastids. In addition they participate in light harvesting during photosynthesis and represent integral components of photosynthetic reaction centers. Carotenoids are the direct precursors of the phytohormone abscisic acid.
Carotenoid biosynthesis as schematically depicted in FIG. 1 has been investigated and the pathway has been elucidated in bacteria, fungi and plants (see for example, Britton, 1988). In plants, carotenoids are formed in plastids.
The early intermediate of the carotenoid biosynthetic pathway is geranylgeranyl diphosphate (GGPP), formed by the enzyme geranylgeranyl diphosphate synthase from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP, see FIG. 1). The subsequent enzymatic step, also representing the first carotenoid-specific reaction, is catalyzed by the enzyme phytoene synthase. The reaction comprises a two-step reaction resulting in a head-to head condensation of two molecules of GGPP to form the first, yet uncoloured carotene product, phytoene (Dogbo et al., 1988, Chamovitz et al., 1991; Linden et al., 1991; Pecker et al., 1992). Phytoene synthase occurs in two forms soluble/inactive and membrane-bound/active and it requires vicinal hydroxyfunctions for activity as present in the surface of plastid galactolipid-containing membranes (Schledz et al., 1996).
While the formation of phytoene is similar in bacteria and plants, the metabolization of phytoene differs pronouncedly. In plants, two gene products operate in a sequential manner to generate the coloured carotene lycopene (Beyer et al., 1989). They are represented by the enzymes phytoene desaturase (PDS, see e.g. Hugueney et al., 1992) and ζ-carotene desaturase (ZDS, see e.g. Albrecht et al., 1996). Each introduces two double bonds yielding ζ-carotene via phytofluene and lycopene via neurosporene, respectively. PDS is believed to be mechanistically linked to a membrane-bound redox chain (Nievelstein et al., 1995) employing plastoquinone (Mayer et al., 1990; Schulz et al., 1993; Norris et al., 1995), while ZDS acts mechanistically in a different way (Albrecht et al., 1996). In plants, the entire pathway seems to involve cis-configured intermediates (Bartley et al., 1999). In contrast, in many bacteria, such as in the genus Erwinia, the entire desaturation sequence forming all four double bonds is achieved by a single gene product (CrtI), converting phytoene to lycopene directly (see e.g. Miawa et al., 1990; Armstrong et al., 1990, Hundle et al., 1994). This type of bacterial desaturase is known not to be susceptible to certain bleaching herbicides which efficiently inhibit plant-type phytoene desaturase.
In plants, two gene products catalyze the cyclization of lycopene, namely α(ε)- and β-lycopene cyclases, forming α(ε)- and β-ionone end-groups, respectively (see e.g. Cunningham et al., 1993; Scolnik and Bartley, 1995, Cunningham et al., 1996). In plants, normally β-carotene carrying two β-ionone end-groups and α-carotene, carrying one α(ε) and one β-ionone end-group are formed.
The formation of the plant xanthophylls is mediated first by two gene products, α- and β-hydroxylases (Masamoto et al., 1998) acting in the position C3 and C3′ of the carotene backbone of α- and β-carotene, respectively. The resulting xanthophylls are named lutein and zeaxanthin.
Further oxygenation reactions are catalyzed by zeaxanthin epoxydase catalyzing the introduction of epoxy-functions in position C5,C6 and C5′,C6′ of the zeaxanthin backbone (Marin et al., 1996). This leads to the formation of antheraxanthin and violaxanthin. The reaction is made reversible by the action of a different gene product, violaxanthin de-epoxydase (Bugos and Yamamoto, 1996).
The gene product leading to the formation of neoxanthin remains to be identified.
Genes and cDNAs coding for carotenoid biosynthesis genes have been cloned from a variety of organisms, ranging from bacteria to plants. Bacterial and cyanobacterial genes include Erwinia herbicola (Application WO91/13078, Armstrong et al., 1990), Erwinia uredovora (Misawa et al., 1990), R. capsulatus (Armstrong et al., 1989), Thermus thermophilus (Hoshino et al., 1993), the cyanobacterium Synechococcus sp. (Genbank accession number X63873), Flavobacterium sp. strain R1534 (Pasamontes et al., 1997). Genes and cDNAs coding for enzymes in the carotenoid biosynthetic pathway in higher plants have been cloned from various sources, including Arabidopsis thaliana, Sinalpis alba, Capsicuin annuum, Naricisstis pseudonarcissus, Lycopersicon esculentum, etc., as can be deduced from the public databases.
Currently relatively little is known about the use of the cloned genes in higher plant transformations and the resulting effects. The expression of phytoene synthase from tomato can affect carotenoid levels in fruit (Bird et al., 1991; Brarley et al., 1992; Fray and Grier-son, 1993).
It has also been reported that constitutive expression of a phytoene synthase in transformed tomato plants results in dwarfism, due to redirecting the metabolite GGPP from the gibberellin biosynthetic pathway (Fray et al., 1995). No such problems were noted upon constitutively expressing phytoene synthase from Narcissus pseudonarcissus in rice endosperm (Burkhardt et al., 1997). Erwinia uredovora CrtI, as a bacterial desaturase, is known to function in plants and to confer bleaching-herbicide resistance (Misawa et al., 1993).
Many attempts have been made over the years to alter or enhance carotenoid biosynthetic pathways in various plant tissues such as vegetative tissues or seeds, or in bacteria. See, for example, WO 96/13149, WO 98/06862, WO 98/24300, WO 96/28014, and U.S. Pat. No. 5,618,988. All of these are restricted to the manipulation of pre-existing carotenoid biosynthetic reactions in the cells. Other applications aiming at altering carotenoid biosynthesis in oil-rich seeds are different, since they provide a sink to accommodate an excess of carotenoids formed due to the increase provoked by the transformation.
It is apparent that there is needed a method of transforming plant material in order to yield transformants capable of expressing all enzymes of the carotenoid biosynthesis pathway necessary to produce carotenes and xanthophylls of interest.